Renal Transport Research Articles

Renoprotective Effects of Brown-Strain Flammulina velutipes Singer in Chronic Kidney Disease-Induced Mice Through Modulation of Oxidative Stress and Inflammation and Regulation of Renal Transporters

Cisplatin, widely used in chemotherapy, acts through mechanisms such as oxidative stress to damage the DNA and cause the apoptosis of cancer cells. Although effective, cisplatin treatment is associated with considerable side effects including chronic kidney disease (CKD). Studies on brown-strain Flammulina velutipes Singer (FVB) have shown its significant antioxidant and immunomodulatory effects. High-performance liquid chromatography (HPLC) confirmed that the FVB extract contained gallic acid and quercetin. This study investigated whether FVB extract can improve and protect against cisplatin-induced CKD in mice. C57BL/6 mice were used as an animal model, and CKD was induced through intraperitoneal cisplatin injection. FVB was orally administered to the mice for 14 consecutive days. N-acetylcysteine (NAC) was administered in the positive control group. Organ pathology and serum biochemical analyses were conducted after the mice were sacrificed. Significant dose-dependent differences were discovered in body mass, kidney mass, histopathology, renal function, inflammatory factors, and antioxidant functions among the different groups. FVB extract reduced the severity of cisplatin-induced CKD in pathways related to inflammation, autophagy, apoptosis, fibrosis, oxidative stress, and organic ion transport proteins; FVB extract, thus, displays protective physiological activity in kidney cells. Additionally, orally administered high doses of the FVB extract resulted in significantly superior renal function, inflammatory factors, antioxidative activity, and fibrotic pathways. This study establishes a strategy for future clinical adjunctive therapy using edible-mushroom-derived FVB extract to protect kidney function.

Modulation of blood pressure by dietary potassium and sodium: Sex differences and modeling analysis.

High Na+ intake has been linked to elevations in blood pressure, whereas K+ has the opposite effect. The underlying mechanisms involve complex interactions among renal function, fluid volume, fluid-regulatory hormones, the vasculature, cardiac function, and the autonomic nervous system. These mechanisms are likely moderated by sex, given the known sex differences in blood pressure regulation and the higher prevalence of hypertension in men. The source of these observed sex differences may be traced to organ and tissue levels, given that kidney function, intrarenal renin-angiotensin system components, renal sympathetic nervous activity, and nitric oxide bioavailability all exhibit sex differences. To assess the functional impact of each of these sex differences, we developed sex-specific computational models to simulate whole-body Na+, K+, and fluid homeostasis, and the effects on blood pressure. The models describe the interactions among the renal system, cardiovascular system, gastrointestinal system, renal sympathetic nervous system, and renin-angiotensin-aldosterone system. Model simulations suggest that women's attenuated blood pressure response to hypertensive stimuli, including high Na+ intake, may be largely attributable to the female renal transporter abundance pattern. Additionally, we investigated the causal link between high K+ intake and blood pressure reduction. The models simulate renal response to high K+ intake, including the immediate gastrointestinal feedforward signals to the kidneys to increase K+ excretion, and the longer-term response to decrease proximal fractional Na+ reabsorption and distal K+ reabsorption. With these assumptions, simulations of high K+ intake yielded kaliuresis, natriuresis, and a substantial reduction in blood pressure, even when combined with high Na+ intake.

Detection of renal tubular transporter proteins in canine urinary extracellular vesicles using liquid chromatography-tandem mass spectrometry.

Urinary extracellular vesicles (UEVs) are membranous particles that carry renal tubular transporter proteins. Here, we evaluate whether selected renal tubular transporter proteins can be detected in UEVs isolated from small volume (1-5 mL) canine urine samples of healthy dogs and canine patients with elevated circulating parathyroid hormone (PTH)/PTH-related peptide (PTHrp) concentrations, hypercortisolism, and primary hypoadrenocorticism using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total creatinine content of each urine sample was calculated from urine volume and creatinine concentration. UEVs were isolated by size exclusion chromatography prior to quantification by nanoparticle tracking analysis and proteomic analysis by LC-MS/MS. Group comparisons were made using non-parametric statistics. Aquaporin-2 (AQP2) and the renal sodium/phosphate co-transporter (NPT2A) were detected in UEVs isolated from small volume samples of almost all healthy dogs but were not detected in most dogs with elevated circulating PTH/PTH related peptide (PTHrp) concentrations, hypercortisolism and primary hypoadrenocorticism. Total creatinine content of the urine sample was strongly positively correlated with the number of UEVs (rs = .84, P < .001); thus, total creatinine was used as a surrogate marker of UEV number. In healthy dogs, AQP2 and NPT2A were both detected in samples containing at least 1.7 × 109 UEVs or 24 μmol creatinine, however in non-healthy dogs, AQP2 and NPT2A were not detected in most samples containing up to 6.3 × 109 UEVs or 32 μmol creatinine.

Obesity-related drug transporter expression alterations in human liver and kidneys.

Pathophysiological changes associated with obesity might impact various drug pharmacokinetics (PK) parameters. The liver and kidneys are the primary organs involved in drug clearance, and the function of hepatic and renal transporters is critical to efficient drug elimination (or reabsorption). Considering the impact of an increased BMI on the drug's PK is crucial in directing dosing decisions. Given the critical role of transporters in drug biodisposition, this study investigated how overweight and obesity affect the gene expression of renal and hepatic drug transporters. Human liver and kidney samples were collected post-mortem from 32 to 28 individuals, respectively, which were divided into the control group (lean subjects; 18.5 ≤ BMI < 25kg/m2) and the study group (overweight/obese subjects; BMI ≥ 25kg/m2). Real-time quantitative PCR was performed for the analysis of 84 drug transporters. Our results show significant changes in the expression of genes involved in human transporters, both renal and hepatic. In liver tissue, we found that ABCC4 was up-regulated in overweight/obese subjects. In kidney tissue, up-regulation was only observed for ABCC10, while the other differentially expressed genes were down-regulated: ABCA1, ABCC3, and SLC15A1. The observed alterations may be reflected by the differences in drug PK between lean and obese populations. However, these findings need further evaluation through the proteomic and functional study of these transporters in this patient population.

B-173 Cystine Analysis in Urine Sample Using the Laser Diode Thermal Desorption technique “LDTD”/Mass Spectrometry (Luxon)

Abstract Background Cystine is a polar amino acid found in the urine of healthy individuals; however, a genetic defect in renal transport can lead to cystine crystals and stones. A usual laboratory approach for diagnosing and monitoring cystinuria is to measure cystine concentration in 24-hour urine. The disease is rare, affecting around 1 in every 10,000 live births. The objective of this study was to develop a rapid assay using Tandem Mass Spectrometry methodology with an internal standard of Cystine derivatized in a urine sample, Luxon Ion Source®, Laser Diode Thermal Desorption (LDTD) technology. Methods The Luxon is a sample introduction and ionization source with a coupled laser diode, providing thermal uniformity, precision, accuracy and speed. Thermally desorbed neutral molecules are analyzed by mass spectrometry (MS). Initially, the sample was derivatized to make the cystine thermally vaporize, followed by liquid-liquid extraction assisted by salting out (SALLE). The isotope-labeled internal standard (Cystine-d4), homemade calibration curve and water quality controls were used for quantification. The analysis was performed using a Sciex 6500+ Triple Quad in APCI (Atmospheric Pressure Chemical Ionization) positive mode. The ratio of peak area to internal standard (ISTD) was used to normalize the signal. A linear regression model with 1/X2 weighting was used to obtain the calibration curve equation and determine the correlation coefficient (r2). Parallelism was assessed by comparing the (r2) of calibration curves prepared in water with six curves prepared with biological matrices. Intra- and interassay precision and accuracy were determined by measuring control samples ranging from low to high concentration in five replicates over 3 consecutive days. For urine analysis, aqueous calibration curves in the concentration range of 1 to 100 mg/L (n=3) showed excellent linearity. The following parameters were evaluated for quantitative analysis: linearity, precision, accuracy, carryover and parallelism.​ Results The calibration curve resulted in a linear response across the entire concentration range, the correlation coefficient (r) was 0.99. For each quality control concentration, CVs were &amp;lt;10%, meeting desirable imprecision and precision specifications. The intraassay CVs were 1.0-2.56% and the interassay CVs were 2.15-4.58% for precision, which reflects the agreement between repeated measurements. Precision results showed similar results, intra-assay CVs were 2.23-3.66% and inter-assay CVs were 1.0-2.77% Conclusions The new LDTD-MS/MS method for the determination of urinary Cystine was accepted after verification and validation of quality control parameters. The technique proved to be effective for reliably and quickly quantifying cystine in urinary samples from human patients, being a valuable tool in the diagnosis and prevention of cystine kidney stones. The assay developed, using the Luxon Ion Source®, based on LDTD (Laser Diode Thermal Desorption) technology, proved to be ultrafast for measuring derivatized cystine in urine

Optimizing renal transporter immunodetection: consequences of freeze-thaw during sample preparation.

Renal transporters (cotransporters, channels, and claudins) mediate homeostasis of fluids and electrolytes and are targets of hormonal and therapeutic regulators. Assessing renal transporter abundance with antibody probes by immunoblotting is an essential tool for mechanistic studies. Although journals require authors to demonstrate antibody specificity, there are no consensus guidelines for kidney sample preparation leading to lab-to-lab variability in immunoblot results. In this study, we determined the impact of sample preparation, specifically freeze-thawed (Frozen) versus freshly processed (Fresh) kidneys (female and male rats and mice) on immunoblot signal detection of 15 renal transporters and the impact of protease inhibitors during homogenization. In female Sprague-Dawley rat kidneys homogenized with aprotinin, Na2EDTA, PMSF, and phosphatase inhibitors, immunodetection signals were ∼50% lower in Frozen versus Fresh samples for most transporters. Inclusion of additional inhibitors (Roche cOmplete Protease Inhibitor, "+") only partially increased transporter immunoblot signals to near Fresh levels. In male Sprague-Dawley rats, immunoblot signal density was lower in Frozen+ versus Fresh+ despite additional inhibitors. In C57BL/6 male mice, immunoblot signals from proximal tubule transporters were lower in Frozen versus Fresh by ∼25-50% and greater in Frozen+. In contrast, immunodetection signal was equivalent in female Frozen+ versus female Fresh+ for claudin 2, villin, AQP1, NKCC2, NCC, ENaCβ, ENaCɣ, claudin 7, AQP2, NKAα1, and NKAβ1. Thus, kidney sample preparation variables, including freeze-thaw and protease inhibition, have substantial transporter-specific effects on quantification of renal transporter abundance by immunoblot. These findings underscore the critical importance of assessing and reporting the impact of sample preparation protocols on transporter recovery to ensure robust rigor and reproducibility. NEW & NOTEWORTHY Freeze-thawing kidneys before homogenization is widely accepted in renal research. This study demonstrates that if kidneys are freeze-thawed just once before homogenization, immunoblot signals are reduced in a transporter-specific manner in rats and mice dependent on sex and that immunoblot signals can be partially recovered by adding additional protease inhibitors. These findings underscore the critical importance of assessing the impact of sample preparation, including freeze-thaw versus fresh, to ensure robust rigor and reproducibility.

The impact of freeze-dried Baiyedancong-Oolong tea aqueous extract containing bioactive compounds on the activities of CYP450 enzymes, the transport capabilities of P-gp and OATs, and transcription levels in mice.

In this study, (-)-epigallocatechin gallate (EGCG) and caffeine extracted from freeze-dried autumn Baiyedancong Oolong tea (FBOT) were orally administered to mice for 7 consecutive days to explore the effects of BOT and its bioactive compounds on the activities and transcription levels of CYP450 enzymes, intestinal effluence transporter P-gp, and renal ingestion Organic Anion Transporters (OATs). Concurrently, EGCG and caffeine enhanced the activities of CYP3A, CYP2E1, and CYP2C37 in the liver of mice, while impairing the transport capabilities of P-gp and OATs. Reduced levels of MDR1 encoding P-gp transcription in the small intestine and renal OAT1 and OAT3 revealed that transcription was involved in the regulation of CYP450, P-gp, and OATs. The reduced transcription level of liver CYP2E1 suggested that CYP2E1 activity may have been elevated due to alternative mechanisms, but not through transcription. The absorption, metabolism, and excretion of drugs may be influenced by the daily consumption or high-dose administration of BOT and its related products, in which EGCG and caffeine may make great contributions.

In vitro and in silico characterization of the transport of selected perfluoroalkyl carboxylic acids and perfluoroalkyl sulfonic acids by human organic anion transporter 1 (OAT1), OAT2 and OAT3

Perfluoroalkyl carboxylic acids (PFCAs) and perfluoroalkyl sulfonic acids (PFSAs) belong to the group of poly- and perfluoroalkyl substances (PFASs), which may accumulate in humans due to their limited excretion. To provide more insight into the active renal excretion potential of PFASs in humans, this work investigated in vitro the transport of three PFCAs (PFHpA, PFOA, PFNA) and three PFSAs (PFBS, PFHxS and PFOS) using OAT1-, OAT2- or OAT3-transduced human embryonic kidney (HEK) cells. Only PFHpA and PFOA showed clear uptake in OAT1-transduced HEK cells, while no transport was observed for PFASs in OAT2-transduced HEK cells. In OAT3-transduced HEK cells only PFHpA, PFOA, PFNA, and PFHxS showed clear uptake. To study the interaction with the transporters, molecular docking and dynamics simulation were performed for PFHpA and PFHxS, for which a relatively short and long half-life in humans has been reported, respectively. Docking analyses could not always distinguish the in vitro transported from the non-transported PFASs (PFHpA vs. PFHxS), whereas molecular dynamic simulations could, as only a stable interaction of the PFAS with the inner part of transporter mouth was detected for those that were transported in vitro (PFHpA with OAT1, none with OAT2, and PFHpA and PFHxS with OAT3). Altogether, this study presents in vitro and in silico insight with respect to the selected PFASs transport by the human renal secretory transporters OAT1, OAT2, and OAT3, which provides further understanding about the differences between the capability of PFAS congeners to accumulate in humans.

Physiological changes in the regulation of calcium and phosphorus utilization that occur after the onset of egg production in commercial laying hens.

At the onset of egg production, physiological changes governing calcium and phosphorus utilization must occur to meet demands for medullary bone formation and eggshell mineralization. The objective of this study was to identify these changes and determine if they are influenced by dietary supplementation with 1α-hydroxycholecalciferol (AlphaD3™, Iluma Alliance). Commercial laying hens fed either a control or AlphaD3-supplemented diet beginning at 18 weeks of age were sampled at 18 (n = 8) and 31 weeks (n = 8/diet) to evaluate mRNA expression associated with calcium and phosphorus utilization in kidney, shell gland, ileum, and liver, circulating vitamin D3 metabolites, and bone quality parameters in humerus, tibia, and keel bone. Though diet did not heavily influence gene expression at 31 weeks, several significant differences were observed between 18- and 31-week-old hens. Heightened sensitivity to hormones regulating calcium and phosphorus homeostasis was observed at 31 weeks, indicated by increased parathyroid hormone receptor 1, calcium-sensing receptor, calcitonin receptor, and fibroblast growth factor 23 receptors in several tissues. Increased renal expression of 25-hydroxylase and vitamin D binding protein ( DBP ) at 31 weeks suggests kidney participates in local vitamin D3 25-hydroxylation and DBP synthesis after egg production begins. Biologically active 1,25(OH)2D3 was higher at 31 weeks, with correspondingly lower inactive 24,25(OH)2D3. Increased expression of plasma membrane calcium ATPase 1 and calbindin in kidney, shell gland, and ileum suggests these are key facilitators of calcium uptake. Elevated renal inorganic phosphorus transporter 1 and 2 and sodium-dependent phosphate transporter IIa at 31 weeks suggests increased phosphorus excretion following hyperphosphatemia due to bone breakdown for eggshell formation. Diet did influence bone quality parameters. Bone mineral density in both humerus and tibia was higher in AlphaD3-supplemented hens at 31 weeks. Tibial bone mineral content increased between 18 and 31 weeks, with AlphaD3-supplemented hens increasing more than control hens. Moreover, control hens exhibited diminished tibial breaking strength at 31 weeks compared to hens at 18 weeks, while AlphaD3-supplemented hens did not. Together, these results indicate supplementation with AlphaD3 enhanced bone mineralization during the medullary bone formation period and elucidate the adaptive pathways regulating calcium and phosphorus utilization after the onset of lay.

Interplay between the Redox System and Renal Tubular Transport.

The kidney plays a critical role in maintaining the homeostasis of body fluid by filtration of metabolic wastes and reabsorption of nutrients. Due to the overload, a vast of energy is required through aerobic metabolism, which inevitably leads to the generation of reactive oxygen species (ROS) in the kidney. Under unstressed conditions, ROS are counteracted by antioxidant systems and maintained at low levels, which are involved in signal transduction and physiological processes. Accumulating evidence indicates that the reduction-oxidation (redox) system interacts with renal tubular transport. Redox imbalance or dysfunction of tubular transport leads to renal disease. Here, we discuss the ROS and antioxidant systems in the kidney and outline the metabolic dysfunction that is a common feature of renal disease. Importantly, we describe the key molecules involved in renal tubular transport and their relationship to the redox system and, finally, summarize the impact of their dysregulation on the pathogenesis and progression of acute and chronic kidney disease.

Distinct and overlapping RGS14 and RGS12 actions regulate NPT2A-mediated phosphate transport

Parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) control serum phosphate levels by downregulating the renal Na-phosphate transporter NPT2A, thereby decreasing phosphate absorption and augmenting urinary excretion. This mechanism requires NHERF1, a PDZ scaffold protein, and is governed by the regulator of G protein signaling-14 (RGS14), which harbors a carboxy-terminal PDZ ligand that binds NHERF1. RGS14 is part of a triad of structurally related RGS proteins that includes RGS12 and RGS10. Like RGS14, RGS12 contains a class 1 PDZ ligand. However, unlike RGS14, the larger RGS12 contains an upstream PDZ-binding domain. The studies outlined here examined and characterized the binding of RGS12 with NHERF1 and NPT2A and its function on hormone-regulated phosphate transport. Immunoblotting experiments revealed RGS12 C-terminal PDZ ligand binding to NHERF1. Further structural analysis disclosed that NPT2A engaged full-length RGS12 and the upstream fragment containing the PDZ domain. Neither the downstream RGS12 portion nor RGS14 interacted with NPT2A. PTH and FGF23 profoundly inhibited phosphate uptake in opossum kidney proximal tubule cells. Transfection with human RGS14, or human RGS12, abolished hormone-sensitive phosphate transport as reported for human proximal tubule cells. RGS12 inhibitory activity resides in the downstream region and is comparable to RGS14. The carboxy-terminal RGS12(667–1447) splice variant is prominently expressed in the kidney and may contribute to regulating hormone-sensitive phosphate transport.

Identification of human-specific amino acid residues governing atenolol transport via organic cation transporter 2

Organic cation transporter 2 (OCT2/SLC22A2) is predominantly localized on the basolateral membranes of renal tubular epithelial cells and plays a crucial role in the renal secretion of various cationic drugs. Although variations in substrate selectivity among renal organic cation transport systems across species have been reported, the characteristics of OCT2 remain unclear. In this study, we demonstrated that atenolol, a β1-selective adrenergic antagonist, is transported almost exclusively by human OCT2, contrasting with OCT2s from other selected species. Using chimeric constructs between human OCT2 (hOCT2) and the highly homologous monkey OCT2 (monOCT2), along with site-directed mutagenesis, we identified non-conserved amino acids Val8, Ala31, Ala34, Tyr222, Tyr245, Ala270, Ile394, and Leu503 as pivotal for hOCT2-mediated atenolol transport. Kinetic analysis revealed that atenolol was transported by hOCT2 with a 12-fold lower affinity than MPP+, a typical OCT2 substrate. The inhibitory effect of atenolol on MPP+ transport was 6200-fold lower than that observed for MPP+ on atenolol transport. Additionally, we observed weaker inhibitory effects on MPP+ transport compared to atenolol transport with ten different OCT2 substrates. Altogether, this study suggests that eight hOCT2-specific amino acids constitute the low-affinity recognition site for atenolol transport, indicating differences in OCT2-mediated drug elimination between humans and highly homologous monkeys. Our findings underscore the importance of understanding species-specific differences in drug transport mechanisms, shedding light on potential variations in drug disposition and aiding in drug development.

Paradoxes in magnesium transport in type 1 Bartter's syndrome and Gitelman's syndrome: a modeling analysis.

Type 1 Bartter's syndrome and Gitelman's syndrome are characterized by mutations in two key renal Na+ transporters, Na-K-2Cl cotransporter (NKCC2) and Na-Cl cotransporter (NCC). Since these two transporters play an important role in regulating magnesium (Mg2+) and calcium (Ca2+) transport in the kidney, significant alterations in the transport of these two electrolytes are observed in type 1 Bartter's syndrome and Gitelman's syndrome. In this study, we used our sex-specific computational models of renal electrolyte transport in rats to understand the complex compensatory mechanisms, in terms of alterations in tubular dimensions and ion transporter activities, that lead to Mg2+ and Ca2+ preservation or wasting in these two genetic disorders. Given the sexual dimorphism in renal transporter patterns, we also assessed how the magnitude of these alterations may differ between males and females. Model simulations showed that in type 1 Bartter's syndrome, nephron adaptations prevent salt wasting and favor Mg2+ preservation but not Ca2+, whereas in Gitelman's syndrome, those adaptations favor Ca2+ preservation over Mg2+. In addition, our models predicted that the compensatory alterations in tubular dimensions and ion transporter activities are stronger in females than in males.NEW & NOTEWORTHY Although changes in Ca2+ excretion in type 1 Bartter's syndrome and Gitelman's syndrome are well understood, Mg2+ excretion displays an interesting paradox. This computational modeling study provides insights into how renal adaptations in these two disorders impact Ca2+ and Mg2+ transport along different nephron segments. Model simulations showed that nephron adaptations favor Mg2+ preservation over Ca2+ in Bartter's syndrome and Ca2+ preservation over Mg2+ in Gitelman's syndrome and are stronger in females than in males.

Abstract P138: Diet Influences the Expression of Renal Sodium-Glucose Cotransporters in Juvenile Male Rats

Poor nutrition and metabolic dysfunction are becoming more prevalent among children, as evident by increasing childhood obesity in recent years. The kidney sodium-glucose cotransporter 2 (SGLT2) is a contributor to overall metabolic homeostasis, with inhibition of SGLT2 significantly reducing circulating glucose and lipids. The study objective was to determine if diet influences the expression of renal glucose transporters in young rats. Weanling male Wistar Kyoto rats received 1) standard laboratory chow, 2) AIN-93G diet, or 3) a high-fat diet (60% kcal, HFD). Each dietary group was then further subdivided to receive either filtered water or 5% (w/v) sucrose solution ad libitum . Four weeks later, animals were euthanized and kidneys were collected and processed. mRNA expression of Sglt1 , Sglt2 , Sqstm1 , and Nlrp3 was determined using gene specific primers and RT-PCR. Renal SGLT2 protein expression was visualized using immunohistochemistry and quantified using Imagej software. Diet significantly influenced the renal expression of both Sglt1 ( P =0.005) and Sglt2 (P=0.046), while beverage did not have a significant effect on either ( P =0.7 and P =0.4, respectively). Rats fed the HFD had more than 30% reduction in Sglt1 mRNA expression as compared to rats fed chow, and ~20% reduction in Sglt2 as compared to rats fed AIN. Preliminary data suggest that diet and beverage significantly altered renal protein expression of SGLT2, with rats fed chow having the lowest renal expression that was slightly increased with sugar consumption (1.5% versus 2.7%, respectively). Rats fed AIN had the greatest expression, but was drastically reduced in rats consuming sugar (4% versus 1.2%, respectively). To determine if autophagy and inflammasome activation is involved, the expression of Sqstm1 and Nlrp3 was measured by RT-PCR. Similarly, rats fed HFD had the lowest expression of both Sqstm1 ( P&lt;0.001) and Nlrp3 ( P=0.03). Sqstm1 and Nlrp3 mRNA expression was decreased with sugar consumption in rats fed chow and AIN, whereas Sqstm1 ( P =0.01) and Nlrp3 ( P &lt;0.001) mRNA expression was increased in rats fed HFD with sugar beverage. Our data suggest that diet significantly influences renal glucose transporter expression in juvenile rats in a way that may be mediated by autophagic impairment. The modulatory effect of sugar consumption on SGLT2 protein, Sqstm1, and Nlrp3 was diet-dependent, highlighting the importance of nutrition to renal physiology within weeks after weaning.

Abstract 37: Targeted Deletion of NKCC1 in Vascular Smooth Muscle Cells Lowers Blood Pressure in Normotensive and Hypertensive Mice

Background: Salt plays a major role in blood pressure homeostasis and the pathogenesis of arterial hypertension. While numerous mechanisms have been described by which Na + can influence arterial blood pressure, the role of Cl - in blood pressure regulation is still largely unexplored. The Na + -K + -2Cl - cotransporter NKCC1 is an important Cl - import pathway in vascular smooth muscle cells (VSMC). To investigate the role of NKCC1 in blood pressure regulation, we generated mice with an inducible VSMC-specific disruption of Slc12a2 . Methods: Mean arterial blood pressure and heart rate were measured by radiotelemetry in unrestrained mice lacking vascular NKCC1 (KO) and wildtype littermates (WT). Cardiac function was assessed by echocardiography. Plasma renin activity and aldosterone concentrations were quantified by radioimmunoassay. Protein expression of renal sodium transporters and channels was analyzed by Western blotting. Results: Mean arterial blood pressure was markedly decreased in Nkcc1 -deficient mice 6 weeks after induction of gene disruption (from 116±2 mm Hg to 103±4 mm Hg, n=18, p&lt;0.001, 2-way ANOVA and Bonferroni post-test), while it did not change in WT control animals (n=17). Heart rate, stroke volume, fractional shortening, and ejection fraction remained unchanged compared to non-induced controls. We did not observe a compensatory increase in plasma renin activity or aldosterone levels, and the blood pressure response to ANG II receptor 1 antagonism by losartan treatment for 7 days was similar in both groups (ΔMAP KO -8.8±3.0 mm Hg, n=8, ΔMAP WT -12.4±1.9 mm Hg, n=7; p&gt;0.05, 2-way ANOVA). In the kidney, the relative expression of total NCC (1.5- fold , n=7-8; p=&lt;0.05, Student’s t-test) and pNCCThr53 (1.9- fold , n=7-8; p=&lt;0.05, Student’s t-test) were modestly increased. The hypotensive effect of Nkcc1 disruption was maintained after induction of hypertension by either infusion of ANG II for 10 days (ΔMAP KO 43.8±6.5 mm Hg, n=6, ΔMAP WT 50.2±7.2 mm Hg, n=4; p&gt;0.05, 2-way ANOVA) or a high salt diet for 10 days (ΔMAP KO 21.7±7.0 mm Hg, n=8, ΔMAP WT 34.0±7.3 mm Hg, n=5; p&gt;0.05, 2-way ANOVA). Conclusions: Our findings indicate that NKCC1 in vascular smooth muscle contributes significantly to the maintenance of arterial blood pressure. Inhibition of NKCC1 in VSMC may provide additional blood pressure reduction in resistant hypertension.

Abstract P422: Neuropeptide FF and Its Receptor NPFFR2 Decrease Renal Sodium Excretion and Increase Blood Pressure in Mice

The kidney is critical in the overall regulation of fluid and electrolyte balance and blood pressure. Neuropeptide FF (NPFF), a morphine-modulating peptide, regulates cardiovascular function through its interaction with two receptors, NPFFR1 and NPFFR2. We now report that NPFF and its receptors, mainly NPFFR2, are expressed in the kidney. NPFF (9.25 mmol, 0.5 mL/hr, 0.05 nmol/day, 7 days) chronically infused underneath the renal capsule, decreased renal sodium excretion (UNaV) from 0.68±0.07 mEq/day, n=5 to 0.43±0.06 mEq/day (n=6/group) in conscious C57BL/6 mice. This was accompanied by an increase in systolic blood pressure (SBP, 114.5±5.0 mm Hg, n=4); the infusion of vehicle (saline) did not affect the blood pressure (96.4±3.0 mm Hg, n=4). The renal subcapsular injection of a single dose (10 µg in 100 µL) of NPFF also increased SBP within 15-35 min (NPFF: 105.5±3.44 mm Hg, n=6); saline injection did not affect SBP (82.6±5.65 mm Hg, n=4). RF-9 (10 µg in 100 µL), an antagonist of NPFF receptors prevented the NPFF-mediated increase in SBP (NPFF alone: 105.5±3.44 mm Hg, n=6; RF+NPFF: 88.5±5.90 mm Hg, n=5), whereas RF-9, alone, had no effect (Baseline: 97.6±2.70 mmHg; RF-9: 90.9±4.70 mm Hg, n=5). The renal subcapsular injection of scrambled peptide (10 µg in 100 µL) had no effect on SBP (86.1±1.83 mm Hg, n=4). However, SBP was decreased by the renal subcapsular infusion of Npffr2 siRNA (98±4 vs 113±3 mmHg; P &lt; 0.05; n=3-5/group) in C57Bl/6 mice fed with a high salt diet (4% NaCl). Furthermore, the SBP of conventional germline Npffr2 knockout (KO) mice fed a high salt (4% NaCl) diet was lower than the SBP of wild-type (WT) littermates (WT: 105.6±2.9 mm Hg, n=5; KO: 90.5±5.5 mm Hg, n=4, P &lt; 0.05). By contrast, in mice fed normal salt (0.8% NaCl) diet, SBP was not different between Npffr2 KO mice and WT littermates (WT: 96.5±4.5 mm Hg, n=5; KO: 98.7±11.7 mm Hg, n=4, P &gt; 0.05). Taken together, NPFF and its receptor, NPFFR2, in the kidney cause salt-sensitive hypertension in mice. Uncovering the functional relevance of renal NPFF in the dynamic regulation of renal sodium transport will lead to a better understanding of blood pressure homeostasis.

Abstract P199: Renal kallikrein-1 is not essential in adaptation to an overnight potassium load

Background: Kallikrein-1 is a serine protease expressed in multiple tissues including the kidney, where it is secreted into the urine. Low urinary kallikrein levels are associated with essential hypertension, whereas high levels are associated with primary hyperaldosteronism in humans. Global kallikrein-1 deletion in mice has been shown to lead to hyperkalemia in response to an acute potassium load as well as defective cleavage of gamma-ENaC, suggesting a direct role in distal K + handling. However, it remains unclear whether effects seen with global kallikrein-1 loss are secondary to loss of extrarenal or renal kallikrein, which is primarily expressed in the connecting tubule. Methods: To test the hypothesis that renal kallikrein-1 is necessary for increased K + secretion in response to acute dietary K + load, we generated mice with conditional knockout of kallikrein-1 in the distal tubule (Klk1 flox - Calbindin-Cre, KS-Klk1 KO). Mice were treated with a K + deficient diet (0% K + , LK) for 9.5 days and then placed in metabolic cages for 12hr overnight collection of urine on control (LK) or high K + (5%, OHK) diets. Metabolic parameters in plasma were measured using iSTAT and kidneys were harvested for downstream western blotting. Results: Deletion of kallikrein-1 in KS-Klk1 kidney was confirmed by western blotting and urine kallikrein activity assay. On LK diet, both WT and KS-Klk1 KO mice developed marked hypokalemia (WT 2.83+/-0.23 vs KO 2.88+/-0.15 meq/dL). Following OHK challenge, hyperkalemia developed to similar levels in both groups (WT 7.13+/-0.56 vs KO 7.74+/-0.35). However, KS-Klk1 KO mice with HK challenge exhibited a more pronounced drop in serum [Na + ] compared with WT (WT 142.6+/-1.4 vs KO 136.6+/-1.3, p=0.022) and correspondingly lower serum [Cl - ] (WT 119.0+/-0.62 vs KO 115.9+/-0.96). Gamma-ENaC quantitation by western blot revealed similar ratios of cleaved: uncleaved species between genotypes. No significant changes between genotypes were detected in urine volume or urine Na+/K+ ratios. Conclusions: KS-Klk1 KO mice do not display defective K+ handling or ENaC processing in response to an acute 12 hr overnight high K + diet. However, they do display relative hyponatremia compared with WT. These data may suggest compensatory mechanisms for potassium transport or a direct a role for renal kallikrein-1 in regulation of renal water transport.

Salinomycin, a potent inhibitor of XOD and URAT1, ameliorates hyperuricemic nephropathy by activating NRF2, modulating the gut microbiota, and promoting SCFA production

Long-term hyperuricemia can induce kidney damage, clinically referred to as hyperuricemic nephropathy (HN), which is characterized by renal fibrosis, inflammation, and oxidative stress. However, currently used uric acid-lowering drugs are not capable of protecting the kidneys from damage. Therefore, uric acid-lowering drugs that can also protect the kidneys are urgently needed. In this study, we first discovered that salinomycin, an antibiotic, can regulate uric acid homeostasis and ameliorate kidney damage in mice with HN. Mechanistically, salinomycin inhibited serum and hepatic xanthine oxidase (XOD) activities and downregulated renal urate transporter 1 (URAT1) expression and transport activity, thus exerting uric acid-lowering effects in mice with HN. Furthermore, we found that salinomycin promoted p-NRF2 Ser40 expression, resulting in increased nuclear translocation of NRF2 and activation of NRF2. More importantly, salinomycin affected the gut microbiota and promoted the generation of short-chain fatty acids (SCFAs) in mice with HN. In conclusion, our results revealed that salinomycin maintains uric acid homeostasis and alleviates kidney injury in mice with HN by multiple mechanisms, suggesting that salinomycin might be a desirable candidate for HN treatment in the clinic.

KCNJ16-depleted kidney organoids recapitulate tubulopathy and lipid recovery upon statins treatment

BackgroundThe KCNJ16 gene has been associated with a novel kidney tubulopathy phenotype, viz. disturbed acid–base homeostasis, hypokalemia and altered renal salt transport. KCNJ16 encodes for Kir5.1, which together with Kir4.1 constitutes a potassium channel located at kidney tubular cell basolateral membranes. Preclinical studies provided mechanistic links between Kir5.1 and tubulopathy, however, the disease pathology remains poorly understood. Here, we aimed at generating and characterizing a novel advanced in vitro human kidney model that recapitulates the disease phenotype to investigate further the pathophysiological mechanisms underlying the tubulopathy and potential therapeutic interventions.MethodsWe used CRISPR/Cas9 to generate KCNJ16 mutant (KCNJ16+/− and KCNJ16−/−) cell lines from healthy human induced pluripotent stem cells (iPSC) KCNJ16 control (KCNJ16WT). The iPSCs were differentiated following an optimized protocol into kidney organoids in an air–liquid interface.ResultsKCNJ16-depleted kidney organoids showed transcriptomic and potential functional impairment of key voltage-dependent electrolyte and water-balance transporters. We observed cysts formation, lipid droplet accumulation and fibrosis upon Kir5.1 function loss. Furthermore, a large scale, glutamine tracer flux metabolomics analysis demonstrated that KCNJ16−/− organoids display TCA cycle and lipid metabolism impairments. Drug screening revealed that treatment with statins, particularly the combination of simvastatin and C75, prevented lipid droplet accumulation and collagen-I deposition in KCNJ16−/− kidney organoids.ConclusionsMature kidney organoids represent a relevant in vitro model for investigating the function of Kir5.1. We discovered novel molecular targets for this genetic tubulopathy and identified statins as a potential therapeutic strategy for KCNJ16 defects in the kidney.

The toxicity of cisplatin derives from effects on renal organic ion transporters expression and serum endogenous substance levels

Acute kidney injury (AKI) is a worldwide public health problem with high morbidity and mortality. Cisplatin is a widely used chemotherapeutic agent for treating solid tumors, but the induction of AKI restricts its clinical application. In this study, the effect of cisplatin on the expression of organic ion transporters was investigated through in vivo and in vitro experiments. Targeted metabolomics techniques were used to measure the levels of selected endogenous substances in serum. Transmission electron microscopy was used to observe the microstructure of renal tubular epithelial cells. Our results show that the toxicity of cisplatin on HK-2 cells or HEK-293 cells was time- and dose-dependent. Administration of cisplatin decreased the expression of OAT1/3 and OCT2 and increased the expression of MRP2/4. Mitochondrial damage induced by cisplatin lead to renal tubular epithelial cell injury. In addition, administration of cisplatin resulted in significant changes in endogenous substance levels in serum, including amino acids, carnitine, and fatty acids. These serum amino acids and metabolites (α-aminobutyric acid, proline, and alanine), carnitines (tradecanoylcarnitine, hexanylcarnitine, octanoylcarnitine, 2-methylbutyroylcarnitine, palmitoylcarnitine, and linoleylcarnitine) and fatty acids (9E-tetradecenoic acid) represent endogenous substances with diagnostic potential for cisplatin-induced AKI.